Identification of novel insertion sites in the Ad5 genome That utilize the Ad splicing machinery for therapeutic gene expression

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doi:10.1016/j.ymthe.2005.07.696

Identification of Novel Insertion Sites in the Ad5 Genome That Utilize the Ad Splicing Machinery for Therapeutic Gene Expression Fang Jin, Peter J. Kretschmer, and Terry W. Hermiston* Gene Therapy Research Department, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA *To whom correspondence and reprint requests should be addressed. Fax: +1 510 669 4246. E-mail: [email protected].

Available online 13 September 2005

Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics. In the case of the oncolytic adenovirus (Ad), however, the genomic packaging capacity is constrained. To address this, we explored whether a transposon-based system could identify sites in the viral genome where endogenous Ad promoters could drive transgene expression via splicing and still maintain the replication capacity of the virus. Using GFP as a reporter gene and an E3-deleted Ad genome as a target, we tested three splicing signals. RACE analysis confirmed that gene expression from the GFP-expressing Ads occurs via splicing and traced expression to the Ad major late promoter (MLP). Replacement of the GFP transposon by an equivalent splice acceptor–luciferase expression cassette in the same orientation confirmed that substitute transgenes are also expressed via splicing from the MLP. Interestingly, insertion of the substitute transgene in the opposite orientation also resulted in expression that, in some cases, originated from within the ITR region of the viral genome. In summary, splice acceptor sequences can be used to control transgene expression from endogenous Ad promoters and this represents a genomically economical approach to arming oncolytic Ads. Key Words: adenovirus, cancer, transposon, virotherapy, oncolytic virus, conditionally replicating virus, gene expression, splicing, armed therapeutic viruses, gene therapy

INTRODUCTION The renewed interest in oncolytic viruses has been sparked by an improved understanding of cancer and virus biology, improved methods to manipulate viral genomes to convey tumor selectivity, and the recognition that standard treatments (chemotherapy, radiation, and surgery) are not curative for the majority of patients with metastatic disease. Consequently, alternative therapies are needed and first-generation oncolytic agents have now entered the clinic in controlled studies that have demonstrated the safety of these agents but also revealed that these initial agents lack effectiveness as monotherapies. The next generation of oncolytic viruses will need to address the issue of potency. In the oncolytic adenovirus (Ad) field, several approaches are being taken, including methods to increase the efficiency of cell lysis [1–7] to increase infectivity (reviewed in [8]) and to barmQ the virus with therapeutic transgenes [9,10]. However, for most viruses, including Ad, the sites available for transgene insertion have been generally restricted to regions known to be nonessential for viral replication in vitro and as such

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require extensive knowledge of the genomic sequence and biology of each candidate virus. As new viruses are identified as oncolytic agents (for example, non-group C Ads), this knowledge will likely not be available. Consequently, a goal of our laboratory is to develop generic methods to scan viral genomes for novel insertion sites that do not adversely affect viral replication. We have previously demonstrated that a transposonbased promoter/transgene expression system could be used to scan the Ad genome to identify novel therapeutic transgene insertion sites for promoter-based transgene expression [11]. In viruses like Ad in which the genomic packaging capacity for therapeutic factors is constrained, methods that reduce the size of the therapeutic expression cassette are needed. One approach to address this need would be to eliminate exogenous promoters by utilizing the endogenous promoters and transcriptional machinery of the virus. This has been done previously in the Ad system in which selective substitution of endogenous genes with therapeutic genes was exploited to generate a system in which therapeutic gene expression levels and

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timing could be predicted based on the expression kinetics of the substituted endogenous gene [12–15]. However, this system was developed based on an understanding of the viral genome and viral life cycle, factors that may not be available in next generation oncolytic viruses. An alternative approach to exploiting the native transcriptional machinery of the virus would be to utilize the highly developed splicing machinery of the human Ad to drive therapeutic gene expression. In this article, we demonstrate the feasibility of using a transposon-based scanning system to identify unique sites for splice-based transgene expression in the replicating Ad. By altering a promoter-based transposon screening system [11] and using GFP as a reporter gene, three splicing signals were tested: (i) a simple splice acceptor, (ii) a splice acceptor plus polypyrimidine tract, and (iii) a consensus splice signal that includes branchpoint, polypyrimidine tract, and splice acceptor sequence. The resultant insertion sites of the splice-dependent GFP-expressing viruses were mapped. Their dependence on splicing and the endogenous promoter responsible for expression were confirmed, and the compatibility of the sites for substitutions was tested. In summary, we have extended the utility of our earlier transposon-based viral arming technology, validating that splice acceptor sequences, rather than an exogenous promoter, can be used to control gene expression in a time-dependent fashion. Using this technology, we were able to define sites where gene expression was restricted to late times postinfection and suggest that the ability to dictate timing of therapeutic gene expression within the viral life cycle will strengthen the versatility and potency of oncolytic viral vectors.

RESULTS Construction and Transposition of Splice Acceptor Transposons We previously developed a transposon-based, nonprejudiced scanning system that enabled identification of novel viral insertion sites for exogenous promoter-directed gene expression that did not interfere with viral replication [11]. To extend the utility of this technology, we explored its ability to enable expression of genes via various splicing signals immediately upstream of a consensus Kozak sequence (reviewed in [16]) and GFP gene. The three splicing sequences utilized were the simplest splice acceptor sequence, CAGG (which we call SSA); the SSA plus polypyrimidine tract (SA); and a third and putatively strongest splice signal composed of the SA plus a branchpoint (BPS) (Fig. 1A, reviewed in [17]). We transposed transposons containing the splice acceptor/ GFP cassettes in vitro to an E3-deleted Ad5 genome within a plasmid and transfected these plasmids into HEK293 cells to generate infectious virus as previously described [11]. We isolated green plaques resulting from the transposition of the GFP transposons to the Ad genome and

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amplified them through A549 cells to obtain viral stocks for subsequent analysis (see Materials and Methods). While only one SSA transposon-containing Ad was isolated, multiple SA and BPS transposon-containing Ad viruses were generated. For each of 14 viruses characterized in detail, we determined the precise nucleotide insertion site (Table 1) as described previously [11], and the direction of transcription and general location in the viral genome are shown in Fig. 1B. While the BPS insertion sites were located in a rather narrow region of the Ad genome in and around the E4 region, the SA insertion sites were more scattered, although confined (except for SA/ PL16) to the rightward ~15% of the Ad genome. It is striking that all transposons were inserted in the same transcription orientation, indicating transcription of the GFP gene from left to right relative to the Ad genome map. While we isolated viruses expressing the GFP gene, it was not clear whether the insertion of the transposon and expression of the GFP gene altered the capacity of the virus to infect cells productively. To test this, we subjected a number of the GFP transposon-containing viruses to an MTS assay. The MTS assay measures the ability of the virus to replicate and spread. As such, direct (e.g., insertions that compromise viral protein function) or indirect (e.g., insertions that alter splicing to, or alter expression of, viral proteins) changes that alter the viral life cycle can be easily, though not specifically, identified. The results are shown in Fig. 1C. As can be seen from these data, some insertion sites (e.g., PL32, PL16) had an adverse effect on the life cycle of the virus compared to the parental CJ51 virus. For PL16, this attenuation is significant and may be due to the insertion of the transposon into the coding sequence of protein V. Protein V plays a major role in the delivery of viral DNA to the host cell and to the redistribution of the major nucleolar proteins nucleolin and B23 to the cytoplasm during the viral infection [18,19]. However, the majority of insertions had no significant effect on viral potency. Kinetic Characterization and Promoter Mapping of Splice Acceptor-Based Gene Expression To begin to characterize the GFP expression from the isolated viruses, we asked whether the GFP gene was expressed early or late in the adenoviral life cycle by analyzing the effects of cytosine arabinoside (araC) on expression of the GFP gene. AraC prevents replication of viral DNA, which in turn is necessary for expression of the adenoviral late genes. We infected A549 cells with the individual viruses, added araC 1 h later, and monitored GFP expression qualitatively over time compared to infection without araC addition and to a previously isolated virus [11] that expresses the GFP gene from the constitutive SV40 promoter. The results from a panel of viruses representing each class of splice acceptor-driven GFP (SSA, SA, or BPS) are illustrated in Fig. 2A. As can be seen, araC had no effect on the expression of GFP

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TABLE 1: Locations of permissible Ad5 insertion sites of splice acceptor transposons Transposon SSA SA SA SA SA SA SA SA SA BPS BPS BPS BPS BPS

Virus name

Insertion region

Insertion site

Ad5/SSA/GFP/PL1 Ad5/SA/GFP/PL8 Ad5/SA/GFP/PL14 Ad5/SA/GFP/PL16 Ad5/SA/GFP/PL17 Ad5/SA/GFP/PL19 Ad5/SA/GFP/PL24 Ad5/SA/GFP/PL28 Ad5/SA/GFP/PL34 Ad5/BPS/GFP/PL2 Ad5/BPS/GFP/PL14 Ad5/BPS/GFP/PL15 Ad5/BPS/GFP/PL22 Ad5/BPS/GFP/PL32

Between E3 14.7K TAA and L5 ATG Between E3 14.7K TAA and L5 ATG Between E3 14.7K TAA and L5 ATG In polypeptide V reading frame 10 bp before the E3 14.7K TAA Between stop codons of L5 and E4 Right in front of E3 14.7K TAA Between E3 14.7K TAA and L5 ATG Between end of ITR and cap site of E4 mRNA Between stop codons of L5 and E4 141 bp after L5 TAA or in E4 ORF6/7 coding region 350 bp after L5 TAA or in E4 ORF6/7 coding region 233 bp after L5 TAA or in E4 ORF6/7 coding region 2 bp before E4 ORF3 ATG codon

C(30863 bp)–T(30864 bp) C(30867 bp)–T(30868 bp) C(30975 bp)–T(30976 bp) C(16643 bp)–T(16644 bp) T(30826 bp)–T(30827 bp) T(32808 bp)–C(32809 bp) C(30846 bp)–T(30847 bp) C(30867 bp)–T(30868 bp) T(35772 bp)–C(35773 bp) C(32809 bp)–A(32810 bp) T(32928 bp)–A(32929 bp) C(33138 bp)–A(33139 bp) T(33026 bp)–T(33027 bp) A(34705 bp)–T(34706 bp)

Numbers refer to Ad genome number [35].

following infection of the SV40/GFP control virus. With the SSA transposon-derived virus, the very faint expression seen without araC was diminished in the presence of araC. Interestingly, araC addition effectively inhibited expression of GFP in the SA and BPS transposon-derived viruses. Similar araC analysis of approximately 100 additional SA or BPS viruses revealed the same pattern of GFP analysis in the presence of araC (data not shown). These results for the SSA, SA, and BPS viruses provide initial evidence that GFP gene expression is controlled by an Ad late promoter. To define the promoter(s) driving expression of representative splice acceptor transposons, we conducted RACE analysis on the GFP transcripts generated by the Ad5/BPS/ GFP/PL2, Ad5/SA/GFP/PL8, Ad5/SA/GFP/PL34, and Ad5/ SSA/GFP/PL1 viruses. We subjected virally infected A549 cell mRNA isolated at 48 h postinfection to RACE/PCR analysis (see Materials and Methods) and cloned and sequenced multiple PCR bands seen following agarose gel analysis. The results of these studies are illustrated in Fig. 2B. The results indicate that the GFP-containing mRNAs derived from Ad5/BPS/GFP/PL2 are limited and that within the transposon region, only the constructed BPS splice acceptor site immediately upstream of the GFP gene is utilized. However, we detected various transcripts for Ad5/SA/GFP/PL8 and Ad5/SA/GFP/PL34 and within

the transposon a variety of splice acceptor sites are recognized. Analysis of additional SA/GFP and BPS/GFP transposon viruses (SA/PL16, SA/PL19, BPS/PL22) yielded similar patterns of splicing (results not shown), suggesting that in general, BPS/GFP transposons yield very btightQ splicing patterns, while SA/GFP transposons have less tight splicing patterns, particularly in the transposon region. The splicing pattern of the one isolated SSA virus (Ad5/SSA/GFP/PL1) is similar to that of Ad5/SA/GFP/PL8 except that no RACE clone indicated precise splicing at the SSA sequence (Fig. 2B). Three of five RACE clones from SSA1 indicated short nonspliced forms of RNA possibly arising from cryptic promoters, with two appearing to originate from within the transposon itself. It is possible that the weak GFP expression observed results from one or more of these mRNA molecules derived by cryptic promoters or from the remaining two splice forms identified. Due to its weak expression and poor splicing pattern, we did not study the Ad5/SSA/ GFP/PL1 virus further. Replacement of the GFP Transposons with Splice Acceptor/Luciferase Expression Cassettes To demonstrate the utility of the identified transposon insertion sites for cloning of relevant transgenes we attempted to replace the GFP-containing transposons

FIG. 1. Characterization of splice acceptor/GFP transposon Ad5 viruses. (A) A schematic representation of the splice transposon is depicted. The Tn7-based transposon system previously described [11] was modified (see Materials and Methods) to make expression of the GFP gene dependent on splicing from an Ad promoter (Ad Pro)-directed mRNA (crosshatched box). The three different splice acceptor sequences, BPS, SA, and SSA, were placed upstream of the GFP gene and are shown with the branchpoint and polypyrimidine sequences. The conserved AG residues at the 3Vend of the intron are underlined. CMR indicates the position of the chloramphenicol-resistance gene within the intron. Splice sites are indicated by ss. (B) Transposon insertion sites in the Ad5 genome. The top is a schematic representation of the Ad5 genome showing selected transposition units and their transcript directions (arrows). Shaded boxes indicate early genes, open boxes indicate late genes. The circled regions are expanded below to indicate transposon insertion sites in more detail (Ad5 genome not to scale). Vertical arrows indicate locations of insertion sites for the labeled viruses, with horizontal arrows indicating the transcription direction of the GFP gene within the transposon. Note that the E3 gene is mostly deleted (see Materials and Methods for details) in the Ad5 used in this study [11]. (C) Potency of transposoncontaining Ad5 viruses compared to the parental virus. MTS assays (see Materials and Methods) were performed on HT29 cells with splice acceptor/GFP transposon-containing clones and the parental Ad5 virus, AdCJ51. Shown are the potency curves for the parental AdCJ51 virus, Ad5/SA/GFP/PL16, Ad5/BPS/ GFP/PL32, Ad5/SA/GFP/PL34, Ad5/BPS/GFP/PL22, Ad5/SA/GFP/PL8, Ad5/BPS/GFP/PL2, and Ad5/SSA/GFP/PL1.

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FIG. 2. Characterization of GFP expression from Ad genomes containing the GFP transposon. (A) Qualitative effects of araC on GFP expression following infection of A549 cells—for details, see Materials and Methods. AraC was added 1 h postinfection, and GFP expression was recorded by microscopy at 48 h. Top left two images—GFP expression in the absence ( araC) and presence (+araC) of araC following infection by Ad5/SV40/GFP/PL29, a virus containing a transposon in which constitutive GFP expression is under control of the SV40 promoter [11]. The remaining images indicate the effects on GFP expression of araC following infection of three representative viruses containing the GFP gene expression under control of splicing to the SSA, SA, and BPS splice transposons. (B) RACE analysis of selected Ad viruses containing different transposon insertions. For each virus, individual lines represent splicing indicated by their different RACE clones (see Materials and Methods). Blue boxes indicate recognized exon regions on the Ad genome—those labeled 1, 2, and 3 indicate the tripartite leader region of the Ad major late promoter-controlled late Ad mRNA. Numbers under virus genomes refer to Ad5 genome numbers in base pairs [35] and known 3Vsplice acceptor (3Vss) or 5Vsplice donor (5Vss) sites are indicated, where applicable. PmeI restriction sites indicate the ends of the transposons, with the light green boxes indicating the BPS, SA, or SSA 3Vsplice acceptor sequences. Arrows above virus names indicate direction of GFP transcription relative to the Ad5 genome. Short arrows above Ad genome (for Ad5/SSA/GFP/PL1) represent RACE clones with no splicing. Ad5 genomes are not drawn to scale.

with a firefly luciferase gene (Luc) preceded by either an SA or a BPS splice site. Due to the transposon design in which blunt-ended PmeI sites are located at the very ends of the transposon, the luciferase substitutions could occur in either orientation [11]. We constructed PmeIdigested BPS or SA splice acceptor/Luc cassettes (see Materials and Methods) and ligated them into PmeI-

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digested DNA of BPS viruses (PL2 and PL22) and SA viruses (PL8 and PL34), followed by transfection into HEK293 cells. We isolated nongreen plaques and analyzed them for their ability to produce luciferase in A549 cells. Table 2 shows the number of luciferase-positive virus plaques and insertion orientations isolated for each

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original transposon insertion. Success in isolation of all four possible derivative viruses (containing the SA/Luc and BPS/Luc cassettes in either direction) for each of the four original transposon viruses varied depending on the position of the transposon insertion site and on the splice acceptor sequences. A number of observations can be made from these experiments. First, it was more difficult to isolate luciferase viruses from the original BPS virus, PL22. Second, isolating luciferase viruses in which the BPS/Luc cassette was in the left-to-right orientation (the same orientation as the original GFP transcription) was more difficult than isolating viruses with this cassette in the reverse orientation. Third, and of particular interest, is that splice acceptor/Luc cassettes can be expressed when inserted in the reverse orientation (compared to the original transposon direction) at a variety of insertion sites. Effects of Luciferase Cassette Substitution on Viral Potency Compared to the Parental GFP Transposon-Containing Viruses To characterize further the splice-based arming technology, we examined whether the replacement of the splice acceptor GFP-transposons with a transgene preceded by a splice acceptor sequence adversely affected the potency of the virus. Therefore we compared in an MTS assay the replication ability of the parental GFP/PL8 transposon virus and its various luciferase derivatives. As can be seen in Fig. 3, replacing the GFP transposon with SA or BPS luciferase expression cassettes in either orientation had no effect on viral potency. Therefore, it is possible to substitute the splice acceptor/GFP transposons with a splice acceptor/transgene cassette without adversely affecting potency. RACE Analysis of Splice-Controlled Luciferase-Expressing Viruses To track the origin of luciferase gene expression, we performed RACE analysis of three luciferase viruses (PL2, PL8, PL34) that share the transgene insertion in the same orientation as in their parent GFP Ad genome. As seen in Fig. 4A, the splicing patterns observed were

TABLE 2: Replacement of transposons with splice acceptor/ luciferase cassettes

Y

Inserted cassette BPS/Luc SA/Luc Y p Y p

Ad5/SA/GFP/PL8 Ad5/SA/GFP/PL34 Ad5/BPS/GFP/PL2 Ad5/BPS/GFP/PL22

1 3 0 0

Parental virus

7 9 6 0

0 4 2 0

2 1 4 1

Arrows indicate orientation of luciferase transcription relative to the Ad5 genome. Numbers indicate the number of viruses isolated containing the luciferase expression cassette.

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FIG. 3. Effect on viral potency by replacement of the GFP transposon with splice-site luciferase gene cassettes. The GFP transposon of Ad5/SA/GFP/PL8 was replaced by luciferase expression cassettes in which the luciferase gene was preceded by a BPS or SA 3Vsplice acceptor site. Potency curves are as follows: AdCJ51 (open circle, blue), Ad5/SA/GFP/PL8 (open rectangle, red), Ad5/SA/PL8/SA/Luc with leftward transcription (LW) (open triangle, green), Ad5/SA/PL8/BPS/Luc (LW) (solid circle, magenta), and Ad5/SA/PL8/BPS/Luc with rightward transcription (solid triangle, brown).

similar for both the SA/Luc and the BPS/Luc viruses and are consistent with the parent GFP viruses (see Fig. 2B) and with transcription originating from the Ad major late promoter (MLP). The one exception to this is a RACE clone indicating transcription initiation 10 bp upstream of the E3 region cap site for the Ad5/SA/PL8/ BPS/Luc virus (Fig. 4A). The agarose gel-isolated DNA band for this RACE clone displayed an intensity similar to that of the other RACE band, suggesting that this band is not an artifact (data not shown). At present we do not have an explanation for this RACE band, other than to propose it may be due to a prematurely terminated reverse transcript of a full-length, MLPderived mRNA. RACE analysis of luciferase viruses in which the transcription orientation was opposite to that of the GFP parent virus generated a greater variety of RACE clones. For the Ad5/SA/PL8/SA/Luc and Ad5/SA/PL8/BPS/ Luc viruses derived from the Ad5/SA/GFP/PL8 parent (Fig. 1B, Table 2), for example, transcription initiated within the E4 transcription unit, with the majority of transcripts initiating from the defined E4 cap site (Fig. 4B). Long (initiating upstream of the E4 cap site at bp 35739) and short (initiating within the E4 transcription unit, bp 31062) RACE clones were also isolated from these luciferase-expressing viruses (Fig. 4B). This suggests that transcription initiation occurs from previously uncharacterized promoters although we cannot rule out that the shorter RACE clones may be due to a lack of extension of reverse-transcribed RNA.

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FIG. 4. RACE analysis of selected viruses in which the original GFP transposon was replaced by expression cassettes in which the luciferase gene is preceded by a BPS or SA 3Vsplice acceptor sequence. Arrows above the virus names indicate the transcription orientation of the 3Vsplice acceptor–luciferase cassette. Further details are as described in the legend to Fig. 2B. 5Vss and 3Vss below the genomes indicate whether those Ad5 genome numbers are known 5Vor 3Vsplice acceptor sites within the E4 transcript. Colored numbers below the genome without a 5Vss or 3Vss designation indicate nucleotide start positions of RACE clones that apparently arise from unspliced (light green) or spliced (dark green) mRNA molecules potentially transcribed from previously unknown promoters or splice sites (see text). The blue arrow (Ad5/SA/PL8/SA/Luc) is a RACE clone starting within the SA sequence. ITR indicates the Ad 3Vinverted terminal repeat. The Ad5 genome is not drawn to scale. (A) BPS and SA luciferase cassettes in the left-to-right transcription orientation following replacement of the transposon in the original Ad5/SA/PL8/GFP, Ad5/ SA/PL34/GFP, and Ad5/BPS/PL2/GFP viruses. (B) BPS and SA luciferase cassettes in the right-to-left transcription orientation following replacement of the transposon in the original Ad5/SA/PL8/GFP, Ad5/SA/PL34/GFP, and Ad5/BPS/PL2/GFP viruses. Nucleotide 35605 is the cap site of the E4 mRNA [35].

RACE analysis of luciferase viruses derived from the parent Ad5/BPS/GFP/PL2 (Fig. 1B, Table 2) generated similar results, with transcription initiating from the E4

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region to generate multiple, spliced mRNA species (Fig. 4B). Similar to the results with the PL8 viruses, transcripts could be detected initiating from the known E4

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cap site at nucleotide 35605. However, unspliced and spliced variants could be detected initiating within the E4 transcription unit for the SA (at nucleotides 33825 and 32967) and BPS (at nucleotide 33825) luciferase variants. In addition, like the PL8 variants, transcripts initiating upstream of the E4 cap site could be detected (nucleotide 35729 and 35721 for the SA and BPS variants, respectively, Fig. 4B). Thus, SA/Luc or BPS/ Luc cassettes inserted at the PL2 and PL8 insertion sites in the right-to-left orientation are driven by the known E4 promoter and from poorly described promoters originating within the E4 transcription unit, which to our knowledge have not been previously described. In contrast to the PL2 and PL8 sites, the PL34 insertion site lies outside of the known Ad transcription units and within the E4 promoter (Fig. 1B, Table 2). Consequently, when the splice/Luc cassette is inserted in the opposite orientation compared to the parent GFP, it is difficult to envision how splice-driven gene expression might occur. Interestingly, expression was detected when the splice/Luc cassette was inserted in the opposite orientation compared to the parent although there is no evidence from the RACE analysis that this was splice mediated. Transcription initiation mapped to nucleotides 35803 and 35864 for the BPS and SA variants, respectively, placing transcription initiation just outside and within the inverted terminal repeat (ITR) region of the virus, respectively (Fig. 4B). We saw additional transcripts beginning just upstream (for Ad5/SA/PL34/SA/Luc) and downstream (Ad5/SA/ PL34/BPS/Luc) of the luciferase gene ATG initiation codon. These results suggest that gene expression was driven at least in part by an ITR-based promoter and/ or enhancer activity and is not the result of splicing.

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Quantitation of Gene Expression Levels from the Luciferase Viruses The insertion sites identified using the splice variant transposon system represent, to our knowledge, unique transgene insertion sites in the Ad5 genome. To characterize these sites further, we sought to measure differences in expression from the various sites and from the different orientations within a given site. Consequently, we measured luciferase expression levels for Ad5/BPS/ GFP/PL2-derived viruses containing the SA/Luc cassette, Ad5/SA/GFP/PL8 viruses containing the BPS/Luc cassette, and Ad5/SA/GFP/PL34-derived viruses containing the BPS/Luc, all with the cassette in both orientations in the given site. To measure the relative strength of expression from these sites, we compared expression levels to those of the strong constitutively active CMV promoter in the virus PL29 [11] in both orientations on the same E3-deleted replicating viral background. As expected (and supported by RACE analysis studies, Figs. 4A and 4B), orientation differences resulted in gene expression generally initiating from two (MLP, E4) different Ad promoters. While one might predict that a single orientation may be consistently superior for all the sites, orientation effects within the Ad genome [11,20], the highly complex nature of the Ad transcriptional machinery (e.g., splicing and polyadenylation signals), and the unusual context of the newly identified gene insertion sites (generally between Ad transcription units or within an Ad promoter) make gene expression levels difficult to predict. Consequently, we examined the effects of orientation differences within a given site on the timing and levels of gene expression over the course of the viral infection (Fig. 5). Several observations can be made. First, gene expression from a given site can vary dramatically,

FIG. 5. Effects of insertion orientation on timing and strength of expression for selected spliceacceptor Ad5 viruses. A549 cells were plated in 24-well plates and after overnight growth were infected with viruses (50 vp/cell); harvested at 8, 16, 24, and 48 h postinfection; and assayed for luciferase activity. RW and LW indicate left-to-right and right-to-left transcription orientation, respectively, of the inserted luciferase gene.

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dependent upon the orientation of the expression cassette and the time point at which gene expression was measured. For example, in the PL8 site, expression from the splice/Luc expression cassette was consistently higher in the leftward orientation. However, this varied significantly over the course of the infection from levels nearly equal at 8 and 48 h postinfection to a difference of slightly over 10-fold at 24 h postinfection (Fig. 5). This is in contrast to expression from the PL34/BPS site, where expression was nearly equal from either orientation up to 24 h postinfection, at which point expression from the rightward orientation dramatically increased (up to 70fold by 48 h postinfection). As reported [11,20] orientation did make a difference in expression from the CMV/ Luc cassette, with the leftward orientation consistently superior throughout the course of the infection. However, this difference was only 3-fold at its peak (48 h postinfection) and therefore does not account for the more significant differences we note above for the PL8 or PL34 sites. Second, significant differences exist in expression levels from the various sites. In the rightward orientation (original orientation of the transposon expression cassette), the highest expression level was derived from the PL34/BPS/Luc site, superior (by nearly 10-fold) to even the CMV/Luc cassette expression levels and nearly 160-fold greater than expression levels derived from the PL2 site at 48 h postinfection. Luciferase expression levels derived from cassettes oriented in the leftward orientation were closer in relative level, with the spread from the highest (PL8) to the lowest (PL34/SA) differing by only slightly over 2-fold and significantly lower than the CMV/Luc cassette (approximately 19-fold less compared to PL8, the highest expression level from the sites where the expression cassette is in this orientation). In summary, gene expression levels and timing of expression must be empirically determined for each newly identified site and orientation within those sites.

DISCUSSION The research described in this paper extends our previous studies utilizing a transposon-based technology for the identification of novel transgene insertion sites that do not adversely affect viral replication and potency [11]. In the current study, we modified the transposon screening system to make gene expression dependent upon splicing and used it to identify unique splice-dependent transgene expression sites compatible with efficient viral replication (Fig. 1, Table 1). AraC analysis of the derived GFPexpressing viruses defined that the unique sites were dependent upon viral replication for the initiation of their expression (Fig. 2A) and RACE analysis mapped the transcripts to the tripartite leader sequence and the Ad MLP promoter (Fig. 2B). None of the identified insertion sites mapped expression to an early promoter. However, it is important to note that the parent virus was deleted of

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most of the E3 region transcription unit. This early region transcription unit is nonessential for viral replication and has been proven to be a site compatible with transgene insertions that are expressed with early gene expression kinetics [12,15]. This region was deleted in these studies to test the capacity of the transposon-based splicing system to identify novel transgene insertions sites. This choice, consequently, may have significantly limited the opportunities to identify sites that express splice-dependent transgenes at early times in the viral life cycle. In addition, we cannot exclude the possibility that our choice of reporter gene, the GFP protein, may also be a contributing factor to our inability to identify splice sites capable of generating transgenes with early expression kinetics. Toxic effects associated with this protein have been previously described [21,22] and thus expression at early times postinfection may be incompatible with the cell or viral life cycle and better tolerated at late times postinfection. Consequently, while we did not identify a site with early expression kinetics, it is possible that the choice of target virus and/or transgene may have been the limiting factor. It is important to note that with the exception of the pIX and IVa2 transcription units, all Ad transcription units encode multiple alternatively spliced mRNAs (reviewed in [23]). The appearance of the alternative splice forms is temporally regulated to ensure that specific proteins are expressed at certain stages and to select levels in the viral life cycle. Consequently, transgene and splice signal insertions, along with the context of the splice signal (e.g., whether preceded by branchpoint and/or polypyrimidine stretch), might be predicted to impact this balance significantly, resulting in either complete ablation of viral replication or compromise of it at some level. The goal of this research, however, was to prove that insertion sites that were dependent upon splicing for expression could be identified and that these insertions would not adversely affect viral replication. While we did see that the insertion sites were limited within the viral genome and that we did compromise the ability of some of the isolated GFP viruses to replicate efficiently (Fig. 1C), the majority of these viruses replicated as efficiently as the parent virus (Fig. 1C). The broader insertion site pattern of the SA-based transposons compared to the more highly clustered BPS-based transposon insertion sites, however, may represent the impact splice signals can make in the viral life cycle. Specifically, all the BPS insertion sites were located outside of the last major late transcription unit, occurring downstream of the last major late transcription unit exon encoding the viral fiber protein L5. Late proteins are essential for the generation of virion coat proteins, efficient maturation of the virion, and packaging of the viral DNA. Consequently, it is possible that the stronger splice signal could not be tolerated upstream of any of the major late transcription unit coding sequences since it could poten-

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tially inhibit or limit splicing to necessary downstream exons, in contrast to the weaker SA splice signal (Fig. 1B). It is important to note that the splice signal alone cannot completely explain the clustering of the BPS sites. A BPS– luciferase substitution could be made into the SA-GFPderived PL8 site (Table 2) and this virus was not altered in its ability to replicate (Fig. 3). This suggests that the transposon (approximately 1.1 kb) and its associated components (including the chloramphenicol gene expression cassette) may also be a contributing factor to site selection. This may also explain why it was difficult to generate either of the splice/Luc cassettes into the PL22 site or the BPS/Luc cassette substitutions into the sites oriented in the same direction (left to right on the Ad5 genome) as the original transposon (Table 2). Splicing is guided by a wide variety of factors, including exonic splicing enhancers, exonic splicing silencers, intronic splicing enhancers (also know as intronic activators of splicing), intronic splicing silencers, and bidirectional splicing enhancers (reviewed in [24,25]), some of which have been identified within the context of the Ad5 genome (reviewed in [23]). The removed transposon material may have had any number of these elements or, alternatively, the combination of insertion site and sequences associated with the luciferase expression cassettes may have created new elements. The ability to isolate SA/Luc cassettes oriented in both directions from both the PL34 and the PL2 GFP viruses, however, suggests that the restriction of BPS/Luc cassette insertions in one orientation does not apply to the SA/Luc cassettes. Further investigation will be necessary to clarify these questions. RACE analysis was conducted on three luciferase viruses in which the orientation was left to right (from PL2, 8, 34) to test whether the splicing pattern could be maintained for a given site. The results were consistent with transcription originating from the MLP (Fig. 4A), in agreement with the RACE analysis of the parental GFP viruses (Fig. 2B). A number of viruses were isolated in which the splice acceptor/Luc cassette was in the reverse orientation compared to the original insertion. Transcription from these viruses was subjected to RACE analysis and the results from the PL2 and PL8 viruses suggest that the majority of RACE clones isolated (from both the SA and the BPS viruses) originated at either the E4 cap site or a site approximately 120 nucleotides upstream of the E4 cap site (Fig. 4B). However, alternative mRNAs were detected that suggested that various less well characterized cryptic promoters from within the E4 transcription unit of the viral genome might also be used. Given that this is RACE analysis, the importance of the different species is not clear and further studies will be needed to answer this question formally. Luciferaseexpressing viruses were also isolated in the reverse orientation for the PL34 virus, which has an insertion site upstream of any known Ad transcription unit. In the

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case of these viruses, RACE analysis indicated that at least some transcription initiation occurred within the right ITR of the Ad virus from where intrinsic promoter and enhancer activities have been described [26–29]. The expression kinetics and levels of gene expression must be empirically defined for each site identified by this process. Outside of promoter strength, factors such as orientation [11,20] and splicing and polyadenylation signals (reviewed in [23,30]) can play a significant role in gene expression in the Ad genome. Our studies on the expression kinetics and levels of gene expression from the various sites (Fig. 5) and orientations within these sites support these previous findings. Importantly, the diversity of expression kinetics and levels should allow investigators to tailor their therapeutic gene to times and levels that best synergize with their therapeutic virus. In this study, we identified sites that were dependent upon splicing and viral DNA replication for the initiation of their gene expression. These properties should allow investigators to link the ability of the virus to distinguish tumor cells from normal cells directly to therapeutic gene expression. For example, oncolytic virus tumor selectivity is generally an engineered or naturally occurring early event in the viral life cycle, preceding viral DNA replication. Viral DNA replication should occur only in the tumor cell and this event should trigger the expression of the therapeutic transgene. Conversely, in the normal cell where the viral life cycle is not allowed to proceed to viral DNA replication, therapeutic transgene expression is inhibited. Consequently, splicing represents a unique opportunity to ensure that therapeutic transgene expression is restricted to the target sites. A splicebased approach for therapeutic gene expression has recently been demonstrated [31], in which the Ad41 long fiber splice acceptor preceded by a poly(A) signal was used to express the yeast cytosine deaminase gene with late kinetics on an oncolytic virus. This selectivity is not available when exogenous, constitutively active promoters (such as CMV) are introduced. It may also be lacking for tissue or tumor selective promoters since these properties could be compromised due to overlapping signaling pathways initiated by viral infection in normal cells and those already present in many cancer cells (reviewed in [32]). In summary, the transposon-based splicing system described in this study represents a genomically economical method for the delivery of a therapeutic transgene from the genomically constrained oncolytic Ad. The scanning feature of the transposon system eliminates the need for extensive biological characterization of the target virus for the identification of sites compatible with the lytic capacity of the virus. Splice-dependent transgene insertion sites create an opportunity to link the natural or engineered tumor selectivity of a virus to efficient, targeted therapeutic expression and represents a significant advancement toward arming conditionally replicat-

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ing viruses to create more clinically viable oncolytic viruses.

MATERIALS

AND

METHODS

Plasmid and adenoviral constructions. SSA, SA, and BPS splice transposon-containing plasmids were constructed as follows. Plasmid pGER54 containing sgGFP [11] was digested with FseI and NheI, and ligated to two pairs of annealed oligos to result in the plasmids pGER99 (SA consensus sequence) and pGER100 (BPS consensus sequence—see Fig. 1A). The two pairs of oligos for the SA sequence were 5V-CCTTTCTCTCTTCAGGCCGCCATGG-3V/5V-CTAGCCATGGCGGCCTGAAGAGAGAAAGGCCGG-3V and for the BPS sequence were 5V-CCTGCTAATCTTCCTTTCTCTCTTCAGGCCGCCATGG-3V/5V-CTAGCCATGGCGGCCTGAAGAGAGAAAGGAAGATTAGCAGGCCGG-3V. Plasmids pGER99 and 100 were digested with FseI and DraIII and ligated to FseI/DraIII-digested pGER57 [11] to result in plasmids pGER111 (containing the SA splice transposon) and pGER112 (containing the BPS splice transposon). For construction of the SSA transposon, pGER111 was digested with SwaI and MluI and the SA splice sequence replaced with the SSA sequence (formed by PCR of SSA/ sgGFP fragment using pGER111 as template with primers 5V-GTACGCTATTTAAATCAGGCCGCCATGGCTAGC-3V and 5V-ATGTCTGACGCGTCTAGTTAGTC-3V and then digested with SwaI and MluI). The resulting plasmid is pGT43. All plasmids except pGER54 were amplified following electroporation to Escherichia coli pir+ cells as described [11] because their R6K origin of replication renders them unable to grow in ordinary lab strains of E. coli lacking the pir gene product necessary for replication and maintenance [33,34]. Construction of plasmid pCJ51 containing a PacIflanked, E3-deleted (starting in the E3 12.5K gene from bp 28138 to bp 30818, 18 bp upstream of the E3 14.7K stop codon), PmeI Ad5 genome has been described [11]. Replacement of transposons in Ad5 with splice acceptor sequence/ reporter gene expression cassettes was as follows. Expression cassettes in which the luciferase gene was preceded by an SA or BPS splice acceptor sequence were PCR amplified with antisense primer 5V-GACGTGTTTAAACTTTACCACATTTGTAGAGGTTTTACTTGC-3Vand sense primer 5VAGCTAGTTTAAACCCTTTCTCTCTTCAGGCCACCATGGAAGA-3V for SA/ Luc or 5V-AGCTAGTTTAAACTGCTAATCTTCCTTTCTCTCT TCAGGCCACCATGGAA-3Vfor BPS/Luc, with plasmid pCMV/Luc [11] as template. The PCR fragments were digested with PmeI and ligated to PmeI-digested HIRTextracted viral DNA (insert:viral DNA molar ratio of 10:1) with T4 ligase (Fast-Link DNA Ligation Kit from Epicentre, Madison, WI, USA). This ligation reaction was then transfected into HEK293 cells using the calcium phosphate method (Invitrogen, Carlsbad, CA, USA) and after 10 days to 2 weeks nongreen viral plaques were isolated and luciferase activity and insertion orientation were determined as described previously [11]. Transposition of splice/GFP transposons to the Ad5 genome and insertion site identification. Methods for in vitro transposition of the splice transposons from pGER111, pGER112, and pGT42 to pCJ51; isolation of virus clones expressing GFP; and identification of transposon insertion sites on the Ad5 genome have been described in detail [11]. Briefly, following in vitro transposition, the reaction mixture was used to transform E. coli cells lacking the pir gene product and plated on chloramphenicol (CM) plates to select for pCJ51 plasmids containing the GFP transposon (which also contains a bacterial CM-resistance marker). Plasmid DNA was extracted from pools of CM-resistant colonies, and following PacI digestion the linearized Ad genomes were used to transfect mammalian cells and green, GFP-expressing viruses were isolated. The precise position of the insertion site in each virus was determined as described [11]. Expression time course and araC analyses. A549 cells were plated in 12(for GFP analysis) or 24-well (for luciferase assay) plates. After overnight culture, the cells were infected with viruses at 100 (for GFP) or 50 vp/cell (for luciferase activity) for 1 h followed by addition of araC (for GFP virus) at a final concentration of 2 AM. AraC levels were refreshed every 12 h. For qualitative analysis of GFP expression, GFP was visualized and photo-

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graphed 48 h postinfection under a Nikon inverted fluorescence microscope (Nikon Diaphot 200; Nelville, NY, USA). For quantitative measurement of luciferase expression, cells were harvested at 8, 16, 24, and 48 h of postinfection, at which times luciferase was measured by the Luciferase Assay System (Promega, Madison, WI, USA). RACE analyses. A549 cells were plated in six-well plates and infected with viruses for 48 h. Total RNA was isolated with an RNeasy Mini kit (Qiagen, Valencia, CA, USA). RACE analysis was performed with BD SMART RACE cDNA amplification kit (BD Biosciences Clontech, Palo Alto, CA, USA). Gene-specific primers were designed with homology to the luciferase gene (5V-CCAACCGAACGGACATTTCGAAGTACTCAGCG-3V) or the sgGFP gene (5V-GGCCATGGAACAGGCAGTTTGCCAGTAGTGC-3V). RACE products were separated on 1% TAE agarose gels and cloned into the pCR2.1 Topo vector (Invitrogen) and sequenced. Viral purification and potency measurements. Mammalian cell lines, viral purification, and potency measurements have been described previously [11].

ACKNOWLEDGMENTS We thank Maxine Bauzon, Paul Harden, Irene Kuhn, Sarasijam Joshi, and the Berlex DNA Sequencing and Cell Sciences groups for reagents and technical assistance and Gabor Rubanyi for critical discussion and review of the manuscript. RECEIVED FOR PUBLICATION MAY 31, 2005; REVISED JULY 28, 2005; ACCEPTED JULY 28, 2005.

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